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NFkB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit (Colorimetric) (ab207216) is a high throughput assay to quantify activation of NFkB (p50, p52, p65, c-Rel, RelB) factors at the same time in one plate. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.
A specific double stranded DNA sequence containing the NFkB consensus binding site (5´ - GGGACTTTCC - 3´) has been immobilized onto a 96-well plate. Active NFkB present in nuclear or whole cell extracts specifically binds to the oligonucleotide. NFkB is detected by primary antibodies that recognizes an epitope on p50, p52, p65, c-Rel or RelB accessible only when NFkB is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout at OD 450 nm. This product detects human, mouse and rat NFkB p65 and RelB, human and mouse p50 and p52, and human c-Rel.
Key performance and benefits:
The transcription factor NFkB is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses. The DNA-binding protein complex recognizes a discrete nucleotide sequence (5´ - GGGACTTTCC - 3´) in the upstream region of a variety of cellular and viral response genes. NFkB is composed of homo- and heterodimeric complexes of members of the Rel (NFkB) family. There are five subunits of the NFkB family in mammals: p50, p65 (RelA), c-Rel, p52 and RelB. These proteins share a conserved 300 amino acid sequence in the N-terminal region, known as the Rel homology domain, that mediates DNA binding, protein dimerization and nuclear localization. This domain is also a target of the IkB inhibitors, which include IkBα, IkBβ, IkBγ, Bcl-3, p105 and p100.
Various dimer combinations of the NFkB subunits have distinct DNA binding specificities and may serve to activate specific sets of genes such as adhesion molecules, immunoreceptors and cytokines. The p50/p65 (NFkB1/RelA) heterodimers and the p50 homodimers are the most common dimers found in the NFkB signaling pathway. In the majority of cells, NFkB exists in an inactive form in the cytoplasm, bound to the inhibitory IkB proteins. Treatment of cells with various inducers results in the phosphorylation, ubiquitination and subsequent degradation of IkB proteins. Proteolytic cleavage of p105 results in two proteins: p50, which has DNA-binding activity but no transactivation domain, and its antagonist, the inhibitory IkBγ protein. This results in the release of NFkB dimers, which subsequently translocate to the nucleus, where they activate appropriate target genes. NFkB can be activated by a number of stimuli, including components of bacterial cell walls, such as lipopolysaccharide, or inflammatory cytokines, such as TNF-α or IL-1β.
|Components||2 x 96 tests|
|10X Antibody Binding Buffer||2 x 2.2ml|
|10X Wash Buffer||1 x 60ml|
|96-well NFkB assay plate||2 units|
|Anti-rabbit HRP-conjugated IgG (0.25 μg/μL)||2 x 11µl|
|Binding Buffer||1 x 10ml|
|Developing Solution||2 x 11ml|
|Dithiothreitol (DTT) (1 M)||1 x 100µl|
|Herring sperm DNA (1 μg/μL)||1 x 100µl|
|Lysis Buffer||1 x 10ml|
|Mutated oligonucleotide (10 pmol/µL)||1 x 100µl|
|NFkB c-Rel antibodies||1 x 11µl|
|NFkB p50 antibodies||1 x 11µl|
|NFkB p52 antibodies||1 x 11µl|
|NFkB p65 antibody||1 x 11µl|
|NFkB RelB antibodies||1 x 11µl|
|Plate sealer||2 units|
|Protease Inhibitor Cocktail||1 x 100µl|
|Raji nuclear extract (2.5 µg/µL)||1 x 40µl|
|Stop Solution||1 x 60ml|
|Wild-type oligonucleotide (10 pmol/µL)||1 x 100µl|
Our Abpromise guarantee covers the use of ab207216 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent concentration.|
Nuclear extracts (10 µg/well) prepared a variety of cell lines were tested for activity of the different NFkB family members. Cell lines were untreated HeLa cells, HeLa cells treated with TNFα, untreated Jurkat cells, Jurkat cells treated with PMA and calcium ionophore (CI), and Raji cells. These results are provided for demonstration only.
ab207216 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"